JP2598024B2 - DNA encoding signal peptide - Google Patents

Description

The present invention relates to a signal peptide having an improved ability to promote protein secretion, and a DNA encoding the same.

As is well known, certain secreted proteins of animal cells are
At its amino terminus, about the mechanism involved in crossing the cell membrane
It is biosynthesized with 15 to 30 peptides, i.e. with a signal peptide. Although the mechanism by which this signal peptide enables the secretory protein to cross the membrane has not yet been completely elucidated, various hypotheses have been proposed to be reliable. According to them, a very small number of non-hydrophobic amino acids contained in the signal peptide first adhere to the cell membrane, and the peptide chain composed of the remaining hydrophobic amino acids penetrates into the cell membrane and then penetrates the membrane It is thought that the secreted protein connected to the protrusion sequentially passes through the membrane. After all secreted proteins have passed through the membrane, the junction between the signal peptide and the secreted protein is cleaved by oxygen present in the membrane, and the mature protein is completed.

When a desired protein is produced in cell culture by genetic manipulation, if the signal peptide is deleted or its function is not sufficient, the expressed protein accumulates in the cell and is degraded by intracellular oxygen. Receiving or insolubilizing it can be extremely difficult to recover it in pure form.

Thus, it will be understood that the signal peptide plays a very important role both in vivo and in the field of biotechnology for producing a desired protein by cell culture.

On the other hand, with the advancement of genetic engineering, it has become possible to produce desired proteins using microorganisms, and there have been remarkable developments in various related technologies, with all aspects of the initial technologies being improved. I have. However, few attempts have been made to increase the secretion efficiency and improve the recovery of the desired protein by using a newly designed signal peptide. The present inventors ligated the DNA encoding the signal peptide of egg white lysozyme and the DNA encoding human lysozyme, incorporated this into an appropriate plasmid, and then introduced this plasmid into yeast to transform human lysozyme. In the process of studying the expression of lysozyme, most of the signal peptide of egg white lysozyme, consisting of 18 amino acid sequences, excluding specific amino acids was replaced with other hydrophobic amino acids, especially leucine (L). It was discovered that the converted product significantly promoted the secretion of human lysozyme, and this was filed for a patent application (Japanese Patent Application No. 62-069764).
No. filed on March 23, 1987).

The present inventors have further studied based on the above findings, and as a result, not only convert most of the 18 amino acid sequences into hydrophobic amino acids, but also the length of the signal peptide amino acid sequence itself by 18 Was found to have a more potent secretagogue-promoting action. The present invention has been completed based on such findings.

In order to explain the present invention more specifically, the 18 amino acid sequences constituting the natural signal peptide of egg white lysozyme are shown below, and the design of the amino acid sequence according to the present invention for promoting secretion is shown below. This will be described in detail.

As understood by the above amino acid sequence formulas, amino acids are described herein primarily in accordance with the one-letter code notation based on the IUPAC-IUB Biochemical Nomenclature Committee Identification Rules. The following are representative amino acids represented by this notation.

M: methionine R ... arginine S ... serine L ... leucine I ... isoleucine V ... valine C ... cysteine P ... proline A ... alanine G glycine F ... phenylaline T ... threonine In doing so, the first and second amino acids (M
And R) and amino acids 16-18 (A, L and G) are believed to be required for attachment to the cell membrane and processing after protein secretion, respectively, so that it is not desirable to modify them. Conceivable. There is some doubt about the substitution of the 13th amino acid (P) with another hydrophobic amino acid, and it cannot be asserted that such substitution is desirable. However, except for the above two points, it was found that the substitution of at least most of the amino acid sequence of this signal peptide with one kind of hydrophobic amino acid, particularly leucine, has a remarkable secretion promoting effect. I knew it. Based on this fact, the present inventors have found that the secretagogue-promoting activity of the signal peptide produced by shortening the length of the hydrophobic region, that is, by reducing the number of leucine by 2, is extremely strong. Was. Therefore, DNA is synthesized so as to encode the signal peptide, and the desired protein,
Especially linked to the gene encoding the secreted protein,
If the host is transformed by incorporating it into an appropriate expression vector, a protein that is extremely easy to recover can be obtained. The present invention has been completed based on such findings, and is a signal peptide consisting of 16 amino acids, wherein the first, second, fourteenth, and sixteenth amino acids M,
It is intended to provide a signal peptide in which all or at least most amino acids except R, A and G are composed of one kind of hydrophobic amino acid. The 14th amino acid in this signal peptide is serine, valine,
Cysteine, threonine and the 16th amino acid may be alanine, serine and cysteine. The present invention also provides a DNA encoding such a signal peptide. The present invention further provides a method for efficiently secreting a target protein out of cultured cells using such a signal peptide.

An example of the amino acid sequence of the signal peptide of the present invention is shown below: The signal peptide shown above is hereinafter referred to as “truncated L-converter” for simplicity.

Hereinafter, the present invention will be described in more detail by taking the case where the secreted protein is human lysozyme and the above-mentioned truncated L-converter is used as the signal peptide as an example.

DNA encoding the improved signal peptide described above
Can be obtained by linking chemically synthesized nucleotide fragments. This DNA may be any DNA that is expressed as a signal peptide that functions in yeast.
Although DNA may be used, in the case of expression using yeast, it is preferable to use codons frequently used in yeast. For more information about codon usage, see The Journal of Biological Chemistry.
iological Chemistry), 257 , 3026-3031 (1982).

The synthesis of the human lysozyme gene has already been published [Chemical and Pharmaceutical Bulletin (Chem. Pharm. Bull.), 34 , 2202 (1986)], and the human lysozyme synthetic gene was obtained accordingly. However, in consideration of the connection with the signal peptide, the present inventors have a Taq I site at the position of the third amino acid F of human lysozyme, and an Xho I site after the stop codon. Encoding human lysozyme partially lacking
A (Taq I-Xho I) was synthesized. In order to quantitatively increase the amount of the synthetic gene thus obtained, it is necessary to first integrate the gene into an appropriate vector and carry out subcloning. As the cloning vector, any vector can be used as long as the gene can be inserted. Specifically, pBR322X in which the Bal I site of the E. coli vector pBR322 was converted to the Xho I site was used.

The synthetic lysozyme gene is incorporated into pBR322X according to a conventional method. Escherichia coli is transformed with the synthetic gene-containing vector thus obtained. The method for transformation of E. coli is known per se, for example, the method of Cohen et al. [Cohe
n, SN et al., Proc. of the National Academy of Sciences (Proc. Natl. Acad. Sci. US
A), 69 , 2110 (1972)].

E. coli 294, E. coli DH1, E. coli W311
0, E. coli C600 and the like can be used.

Next, the plasmid DNA into which the gene has been inserted is subjected to, for example, an alkaline extraction method [Birnboim, HC and Doly, J., Nucleic Acids Res.
7 , 1513 (1979)].
By treating the obtained plasmid DNA with an appropriate restriction oxygen, the inserted gene can be cut out and isolated by, for example, agarose gel electrophoresis or polyacrylamide electrophoresis. These series of operations are known and described in the literature, for example, "Molecular Cloning (1982), Cold Spri
ng Harbor Laboratoy ". The isolated gene is forwardly ligated to the promoter of an appropriate expression vector and downstream of the signal peptide coding region to construct an expression plasmid.

Encodes a signal peptide and human lysozyme
For the synthesis of DNA, a yeast having a high codon usage may be used, but a codon in a lower rank may be used for creating a restriction oxygen recognition site or the like.

Chemical synthesis of DNA can be performed, for example, by the method of Crea et al. [Crea,
R. et al., Proc. Of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA),
75 , 5765 (1978)].

Many expression vectors are already known, and any expression vector may be used as long as it can be actually used for expression of the gene. Specifically, pPHO17, pGLD90
6-1, pcDX [Okayama, H and Berg, P, Molecular
And Cellular Biology (Mol. Cell. Biol.),
3 , 280 (1983)], and pKSV-10 (Pharmacia).

That is, when yeast is used as a host, for example, a PHO5 promoter, a GLD promoter, a PGK promoter, an ADH promoter, a PHO18 promoter are used as promoters, and when an animal cell is used as a host,
As the promoter, for example, SV40 early gene promoter, metallothionein promoter, heat shock promoter and the like can be used.

The use of an enhancer for expression is also effective.

Examples of the host used for expression include yeasts such as Saccharomyces cerevisiae, mouse L cells, Chinese hamster oocytes (CHO), and the like, but other eukaryotic cells may also be used. can do.

When yeast is used as a host, for example, the method of Hinnen et al. [Proceding of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA),
75 , 1927 (1978)].

Transformation methods of eukaryotic cells such as animal cells are known per se, for example, "Protein / Nucleic Acid / Oxygen, Vol. 28, 1983,
"Transfection and expression of recombinant genes into cells" (Kyoritsu Shuppan)
It is performed according to the description of.

A host cell is transformed with the obtained secretion expression plasmid to obtain a desired recombinant. The transformant thus obtained is cultured by a method known per se.

When using yeast, for example, Burkhold
er minimal medium [Bostian, KL et al., Processing of
The National Academy of Science (Pro
Natl. Acad. Sci. USA), 77 , 4505 (1980)]. Culture is usually at 15 ° C to 40 ° C, preferably at 24 ° C to 37 ° C.
The reaction is carried out for 10 to 144 hours, preferably 24 to 96 hours, and if necessary, ventilation or stirring may be added.

When a transformant using a eukaryotic cell such as an animal cell as a host is used, for example, MEM [H.
Eagle, Science, 130 , 432 (1959)],
Dulbecco's Modified Eagle's Medium [Oigad Laub and William J. Rutter, Journal of Biological Chemistry (J. Biol. Chem.), 258 , 6043 (198
3)]. The cultivation is usually performed at 30 to 42 ° C, preferably 35 to 37 ° C for about 1 to 10 days.

After completion of the culture, the cells and the supernatant are separated by a method known per se. Human lysozyme remaining in the cells can be obtained by normal methods in the art, such as ultrasonic crushing, crushing using a French press, mechanical crushing such as trituration, crushing using cell lysis oxygen, etc. Extract after crushing. If necessary, a surfactant such as Triton-X100 or deoxycholate is added to extract the produced human lysozyme. The thus obtained culture supernatant and human lysozyme contained in the cells are separately quantified, and the ratio between the two is compared. The obtained human lysozyme can be purified according to a conventional protein purification method, for example, salting out, isoelectric point precipitation, gel filtration, ion exchange chromatography, high performance liquid chromatography (HPLC, FPLC, etc.).

Hereinafter, the present invention will be specifically illustrated by way of Examples,
Since the signal sequence shown in the examples is very generalized, the protein to be secreted is not limited to human lysozyme.

Example 1 Preparation of Cloning Vector pBR322X Restriction oxygen Bal I was added to E. coli vector pBR322 (5 μg).
(1.5 units), and add 40 µl of the reaction solution [10 mM Tris-
HCl (pH 7.5), 10 mM MgCl ₂ , 1 mM dithiothreitol]
After reacting at 37 ° C. for 5 hours, phenol was extracted as usual, and the DNA was precipitated with ethanol. XhoI linker d [pCCTCGAGG] phosphorylated on this DNA (New England
Biolabs) (50 ng) was added, and both were ligated with T4 DNA ligase according to a conventional method.

Escherichia coli DH1 strain was transformed with this reaction solution, and a plasmid was extracted from the obtained ampicillin-resistant and tetracycline-resistant colonies by an alkaline extraction method (described above).
A plasmid pBR322X in which the site was converted to an Xho I site was obtained.

Example 2 Preparation of Human Lysozyme Gene Fragment Having Taq I Site Near N-Terminus of Human Lysozyme Gene synthesis was performed according to the report of Ikehara et al. (Supra).
However, the nucleotide fragment in the report of Ikehara et al., U
2 (TTGAGAGATGCGAAT) instead of U2 '(CGAGAGATGCGAA
T) instead of L2 (TCTGGCTAATTCGCATC) instead of L2 '(TCTGGC
TAATTCGCATCTCT) to synthesize U2 ', U3-U26, U2', L3-L2
Six fragments were ligated.

As a result, the codon TTT for the third amino acid, phenylalanine (F), became TTC, and unlike the report of Ikehara et al., A gene lacking a part at the 5 'end, which had a new Taq I site in that part. Was obtained (first
Figure).

Example 3 Subcloning of Human Lysozyme Gene Fragment Having Taq I Site Six 2.6 units of restriction oxygen Xho I and 6 units of restriction oxygen Cla I were added to 2.6 μg of plasmid pBR322X constructed in Example 1 to a 35 μ reaction solution [33 mM acetate buffer. In a solution of pH 7.9, 66 mM potassium acetate, 10 mM magnesium acetate, 0.5 mM dithiothreitol, 0.01% BSA] at 37 ° C. for 1 hour, the protein was removed with phenol and precipitated with cold ethanol. The DN
A (200 ng) was mixed with 100 ng of the human lysozyme gene fragment prepared in Example 2, and a 10 μl reaction solution [66 mM Tris-HCl
(PH7.6), 10mM ATP, 10mM spermidine, 100mM MgC
l _2, in 150 mM DTT, 2 mg / ml BSA, 5 units T4DNA ligase, was bound to DNA by the action overnight at 14 ° C.. Using this reaction solution, Escherichia coli DH1 strain was transformed according to the method of Cohen et al. A plasmid was isolated from the obtained transformant by the alkali extraction method (described above), and the molecular weight and the degradation pattern by restriction oxygen were examined. Thus, pLYS221 into which the human lysozyme gene fragment was inserted was obtained. Ec of pLYS221
The oR I-Xho I fragment was separated and its base sequence was determined by the dioxynucleotide synthesis chain termination method.
As shown in the figure, the human lysozyme gene Taq
An I-Xho I fragment was obtained.

This sequence encodes from Glu at position 4 to Val at position 130 in the amino acid sequence of human lysozyme.

Example 4 Preparation of DNA Encoding Truncated L-Converter DNA encoding the truncated L-converter was prepared as a signal sequence for secreting human lysozyme into the medium. The synthesized nucleotide sequence is shown in FIG. 2. An Xho I site is provided at the 5 'end, and a Taq I site containing a human lysozyme coding region is provided at the 3' end. The entire sequence consists of eight oligonucleotides (# 1
~ # 8) and their synthesis was carried out by the phosphoramidite method [Caruthers, MH et al., Tetrahedron Letters (Te
trahedron Letters), 22 , 1859 (1981)].

First, 10 μm (5 μg) of each of fragments # 2 to # 7 shown in FIG. 2 was mixed, and a 10-fold concentration of a kinase buffer [0.5 M Tris-HCl, 0.1 M MgCl ₂ , 0.1 M mercaptoethanol pH7 was added. .6] 20μ, 10mM ATP20μ, T
4 Polynucleotide kinase (Takara Shuzo) 20μ (50
u), 80 μm of distilled water was added and reacted at 37 ° C. for 2 hours, followed by treatment at 65 ° C. for 20 minutes to stop the reaction. Fragments # 1 and # 8 were each added to this reaction solution in an amount of 10 μ (5 μg), and T4 ligase (manufactured by NEB) 10 μ was added, followed by reaction at 14 ° C. overnight. The reaction solution was subjected to 10% polyacrylamide electrophoresis to cut out a 70 bp fragment, and then extracted from the gel by electrophoretic elution. This was dissolved in 45 µ of distilled water, to which 6 µ of a 10-fold concentration of a kinase buffer (see above), 6 µ of 10 mM ATP, and 2 µ (5 µ) of T4 polynucleotide kinase (see above) were added and reacted at 37 ° C for 1 hour. After that, it was stored at -20 ° C.

Example 5 Construction of Secretory Expression Plasmid pLYS221 (236 μg) obtained in Example 3 was treated at 37 ° C. for 2 hours with 120 u of EcoR I (manufactured by Nippon Gene) and 120 u of Xho I (manufactured by Nippon Gene) to obtain a human lysozyme code. The area was cut out. Add this fragment to Taq I
(Nitsupongene) The mixture was treated with 26u at 65 ° C for 1 hour to cut out a human lysozyme coding region partially lacking the N-terminus.

About 1 μg of this fragment was added to the shortened L-
Mix 0.5 μg of DNA encoding the converter signal sequence,
After reaction at 16 ° C. for 16 hours in the presence of 800 u of T4-ligase (described above), the cells were treated with XhoI (42 u).

10 ng of the obtained XhoI fragment and 1 ng of a vector obtained by treating the yeast expression vector pGLD906-1 (Japanese Patent Laid-Open No. 61-43991) with XhoI were mixed, and both were ligated in the presence of T4 ligase.

E. coli DH1 was transformed with the reaction solution as described above,
A number of plasmids were obtained in which the signal sequence coding region and the human lysozyme gene were inserted downstream of the GLD promoter in the same direction as the promoter. One of them is pGE
L.L8 (see FIG. 3) was used for the following experiments.

Example 6 Preparation of yeast transformant Saccharomyc using expression plasmid pGEL.L8 obtained in Example 5
es cerevisiae AH22R ^- transformed with Hinnen et al method (supra), transformant Saccharomyces cerevisiae AH22R
^- / was obtained pGEL · L8. This strain has been deposited with the Research Institute of Microbial Industry, National Institute of Advanced Industrial Science and Technology under the accession number FERM P-9426 (deposit date: June 20, 1987).

Example 7 Culture of transformant Saccharomyces cerevisiae obtained in Example 6
AH22R ^- / pGEL · L8 the Burkholder [(. Amer.J.Bot) American Journal of Botany 30, 206 (1943)]
Modified medium III (0.4 g of KH ₂ PO ₄ , 10 g of glucose,
A test tube containing 5 ml of asparagine (5 g, sucrose 80 g) was inoculated, and cultured with shaking at 30 ° C. for 3 days. 1 ml of the obtained culture was transferred to a test tube containing 4 ml of the above medium III, and cultured at 30 ° C. for 1 day with shaking. 2 ml of this culture solution was transferred to a 200 ml Erlenmeyer flask containing 18 ml of the above medium III, and cultured with shaking at 30 ° C., and assay samples were prepared after 48, 72, 96 and 120 hours.

Example 8 Preparation of Assay Sample The culture solution obtained in Example 7 was centrifuged to separate the supernatant from the cells. The supernatant was subjected to an assay, and the cells were washed with 50 mM phosphate buffer (pH 7.0) containing 1.2 M sucrose, and then the cells were washed with Zymolyase [Seikagaku Corporation].
Was suspended in the same buffer added to 0.5 mg / ml,
The reaction was performed at room temperature for 2 hours. To this reaction solution, 4-fold volume of 50 mM phosphate buffer (pH 7.0) [containing 10 mM EDTA and 1 mM PMSF] was added and allowed to react at room temperature for 1 hour. Then, the supernatant was collected to obtain a cell extract.

Example 9 Measurement of Human Lysozyme Production Amount The supernatant obtained in Example 8 and a cell extract were subjected to a human lysozyme assay.

The measurement of human lysozyme activity is mostly performed using the Worthigton Enzyme Manual, p1
00, Worthigton Biochemical Corporation, USA, 1972)
According to Sigma was used as standard human lysozyme. One unit is Micrococcus luteus (Micrococcusluteus) in 0.1 M phosphate buffer (pH 6.9).
(Manufactured by Seikagaku Corporation) as a substrate was reacted at 25 ° C. for 1 minute, and the amount of oxygen required to reduce the absorption at 450 mμ by 0.001 was determined. The amount of human lysozyme produced by performing the same experiment three times was as shown in Table 1 below. In addition, the result which performed the same experiment as the above using the signal sequence of egg white lysozyme was shown as a target.

As is evident from Table 1, a larger amount of human lysozyme was secreted outside the cells when the truncated L-converter was used than when the signal sequence of egg white lysozyme was used. Significant differences were also seen in the total intracellular and extracellular lysozyme production.

Example 10 Purification of secreted lysozyme Transformant Saccharomyces cerevisiae obtained in Example 6
AH22R ⁻ / pGEL·L8 was inoculated into three test tubes containing 5 ml of the medium described in Example 7, and cultured with shaking at 30 ° C. for 3 days. Five 200 ml Erlenmeyer flasks each containing 18 ml of the above medium were prepared, and 2 ml of the above culture solution was transferred to each of them, followed by shaking culture at 30 ° C. for 1 day. Next, 5 Erlenmeyer flasks containing 200 ml of the above medium
Prepare this and transfer 20 ml of this culture to each,
Cultured for days. This culture was centrifuged to separate the supernatant from the cells. The supernatant (about 1) is adsorbed on a cation exchange resin (CM-Toyopearl 650C) column (1.6 cm × 36 cm) equilibrated with 50 mM sodium phosphate buffer (pH 6.5) and washed with 500 ml of the same buffer. After that, elution was carried out with the same buffer containing 0.5 M NaCl. The eluate was collected in 5 ml portions, and the lysozyme activity of each fraction was measured according to Example 9. 200 from the fraction with the highest lysozyme activity
μ and reverse-phase high-performance liquid chromatography (TSKgel
(ODS120T column). At this time, elution was performed by a linear gradient of 0 to 100% of acetonitrile containing 0.1% trifluoroacetic acid for 30 minutes, and an absorption peak at 280 nm was collected. The solvent of this fraction was evaporated under reduced pressure to obtain a purified human lysozyme sample as a dry powder.

Example 11 Analysis of Purified Lysozyme For the purified human lysozyme sample purified by the above method, the amino acid sequence of the N-terminal 10 residues was determined, and the amino acid analysis was performed. The N-terminal sequence was determined using a purified sample (7 μg) using a protein sequencer (Applied Biosystems).
477A). Amino acid analysis was carried out by treating 20 μg of the purified sample with 0.2 ml of 6N HCl at 110 ° C. for 24 hours to hydrolyze.Then, the reaction solution was dried under reduced pressure, and 0.5 ml of 0.02N
The solution was dissolved in HCl and analyzed by an automatic amino acid analyzer (Hitachi 835). Further, in order to quantify the cysteine residue, 20 μg of the purified sample was separately treated in 0.1 ml of formic acid at 0 ° C. for 2.5 hours, dried under reduced pressure, and treated in the same manner as described above for amino acid analysis.

(A) Determination of N-terminal sequence The results as shown in Table 2 below were obtained.

(B) Composition analysis of amino acids The results shown in Table 3 below were obtained.

The above results revealed that the shortened L-converter functions normally and secretes mature human lysozyme.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing a sequence in which the 5 ′ end of the DNA sequence encoding human lysozyme has been partially deleted. FIG. 2 shows a shortened L-converter signal peptide and the corresponding DN.
FIG. 3 is a schematic diagram showing the A sequence, and FIG. 3 is a construction diagram of human lysozyme secretion expression plasmid pGEL·L8.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical indication C12R 1: 865) (C12P 21/02 C12R 1:91)

Claims (5)

(57) [Claims] 1. The first, second, and second amino acids comprising 16 amino acids.
A signal peptide in which all or at least most amino acids except for the 14th and 16th amino acids M, R, A and G are composed of L (however, the 14th amino acid is S, V, C or T). And the 16th amino acid may be A, S or C). 2. The formula: MRLLLLLLLLLLPLA.
2. The signal peptide according to item 1, consisting of an amino acid sequence represented by -ALG. 3. The method according to claim 1, comprising 16 amino acids, wherein the first, second, and
A signal peptide in which all or at least most amino acids except for the 14th and 16th amino acids M, R, A and G are composed of L (where the 14th amino acid is S, V, C or T (The 16th amino acid may be A, S or C). 4. The formula: MRLLLLLLLLLLPLA.
4. The DNA according to item 3, which encodes the amino acid sequence represented by ALG. 5. The formula: The DNA according to item 4, which is represented by: